Neuromodulation is essential for producing adaptive behaviors in response to changing environmental demands. Modulation by the serotonergic system is important for the control of movement, the root of all behavior. The overall goal of this project is to elucidate the role of the serotonergic system in motor behavior. Spinal motor neurons, which directly control peripheral muscle activity, are densely innervated by brainstem serotonin neurons. Furthermore, electrophysiological studies have established that serotonin is a potent regulator of motor neuron excitability. However, the role of serotonergic modulation in controlling motor output within a behavioral context is unclear. I will use novel genetic and viral approaches in mice to investigate the function of genetically defined serotonergic circuits in motor control. In the K99 Aims, I will examine the function of serotonergic input to spinal motor neurons during locomotor behavior. First, I will systematically dissect the anatomical organization of serotonergic inputs to spinal motor neurons using anterograde and retrograde tracing strategies. Second, I will perform in vivo electrophysiology to test whether serotonin-spinal inputs regulate the gain of synaptic input to motor neurons. Finally, I will test the hypothesis that increased activity of serotonergic neurons during fast locomotion is required for producing the increased muscle activity for vigorous movement. I will perform functional perturbation and imaging studies to determine how serotonergic input to motor neurons affect muscle output and locomotor behavior. I will use chronic electromyography (EMG) recordings from limb muscles during these experiments providing precise readout muscle activity to determine how the serotonergic system adjusts motor output. In the R00 aims, I propose experiments to define the cellular mechanisms by which neuromodulators act upon target neurons to mediate behavioral effects, as well as the context-dependent regulation of neuromodulatory pathways. First, I will dissect the role of metabotropic vs. ionotropic excitatory serotonin receptors in motor neurons during motor behavior. Second, I will test the hypothesis that brain regions controlling locomotion drive activity of serotonin- spinal pathways to facilitate appropriate muscle output for the behavioral context. Together, these experiments and training experience will set the stage for a career in cellular, circuit and behavioral level investigation of genetically-defined neuromodulatory populations in motor control. The proposed studies will provide new insight to the function of serotonin in motor control, beyond the classic physiological and pharmacological approaches used previously by the field. Furthermore, this work aims to inspire new clinical approaches for treatment of disorders or injury of the spinal cord that influence production of movement.